Non-Typhoidal Salmonella in poultry meat and diarrhoeic patients: prevalence, antibiogram, virulotyping, molecular detection and sequencing of class I integrons in multidrug resistant strains

Abstract

Background: The worldwide increase of food-borne infections with antibiotic resistant pathogens constitutes a major public health problem. Therefore, this study aimed to determine the prevalence, antibiogram, virulence genes profiles and integron characteristics of non-typhoidal Salmonella spp. isolated from poultry meat and diarrhoeic patients in Egypt.

Methods: A total of 150 samples comprising (100 poultry meat and 50 diarrhoeic patients' stool) were examined for the presence of Salmonella spp. using culture methods followed by biochemical and serological identification of the isolates. All Salmonella strains were tested for their susceptibility to the antibiotics using disk diffusion method and screened for the presence of virulence genes and class I integrons using PCR.

Results: The overall prevalence of Salmonella spp. in poultry meat samples was 10 % compared to 4 % in diarrhoeic patients. All the isolates were serologically identified into Salmonella Typhimurium (seven isolates), S. Derby, S. Kiel, S. Rubislaw (one isolate, each) and untypable strains (two isolates). Antibiotic susceptibility testing showed a higher resistance of the total isolates to erythromycin and tetracycline (100 %, each), followed by amoxicillin-clavulanic acid (91.7 %), trimethoprim-sulfamethoxazole (83.3 %), streptomycin, nalidixic acid, ampicillin-sulbactam (75 %, each), gentamycin, ampicillin (66.7 %, each), chloramphenicol (58.3 %), ciprofloxacin (25 %) and ceftriaxone (16.7 %). Virulence genes profiles revealed the presence of sopB gene in five Salmonella strains isolated from poultry meat (n = 3) and humans (n = 2). Moreover, pefA was only identified in three isolates from poultry meat. On the other hand, S. Kiel and S. Typhimurium (one isolate, each) were harboring hilA and stn genes, respectively. Class 1 integrons were detected in all Salmonella spp. with variable amplicon sizes ranged from 650-3000 bp. Sequencing of these amplicons revealed the presence of gene cassettes harboring aac(3)-Id, aadA2, aadA4, aadA7, sat, dfrA15, lnuF and estX resistance genes. Nucleotide sequence analysis showed point mutations in the aac(3)-Id of S. Derby, aadA2, estX-sat genes of S. Typhimurium. Meanwhile, frame shift mutation was observed in aadA7 genes of S. Typhimurium.

Conclusions: Increasing rate of antimicrobial resistance and class 1 integrons among multidrug resistant Salmonella spp. has prompted calls for the reduction of antimicrobial use in livestock to prevent future emergence of resistance.

Keywords: Class I integrons; Salmonella spp.; Virulence; Zoonoses, multidrug resistance.

Fig. 1

Agarose gel electrophoresis showing an amplification of pefA gene (700 bp) in Salmonella isolates from poultry meat and humans. Lane N: negative control (pefA⁻ Salmonella strain), lane L: DNA ladder (100 bp), lane P: positive control (pefA⁺ Salmonella strain), lane 1: pefA⁻ (S. Derby, duck), lane 2: pefA⁻ (S. Kiel, chicken), lane3: pefA⁺ (untypable, duck), lane 4: pefA⁺ (S. Rubislaw, chicken), lanes 5,6, 10, 11: pefA⁻ (S. Typhimurium, chicken), lane 7: pefA⁻ (untypable, duck), lanes 8,9: pefA⁻ (S. Typhimurium, humans), lane12: pefA⁺ (S. Typhimurium, chicken)

Fig. 2

Agarose gel electrophoresis showing an amplification of hilA gene (854 bp) in Salmonella isolates from poultry meat and humans. Lane N: negative control (hilA ⁻ Salmonella strain), lane L: DNA ladder (100 bp), lane P: Positive control (hilA⁺ Salmonella strain), lane 1: hilA ⁻ (S. Derby, duck), lane 2: hilA⁺ (S. Kiel, chicken), lane 3,7: hilA⁻ (untypable, duck), lane 4: hilA⁻ (S. Rubislaw, chicken), lanes 5, 6, 10, 11,12: hilA⁻ (S. Typhimurium, chicken), lanes 8,9: hilA⁻ (S. Typhimurium, humans)

Fig. 3

Agarose gel electrophoresis showing an amplification of sopB gene (517 bp) in Salmonella isolates from poultry meat and humans. Lane N: negative control (sopB⁻ Salmonella strain), lane L: DNA ladder (100 bp), lane P: Positive control (sopB⁺ Salmonella strain), lane 1: sopB⁻ (S.Derby, duck), lane 2: sopB⁻ (S. Kiel, chicken), lanes 3, 7: sopB⁺ (untypable, duck), lane 4: sopB⁻ (S. Rubislaw, chicken), lanes 5: sopB⁻ (S. Typhimurium, chicken), lane 6: sopB⁺ (S. Typhimurium, chicken), lanes 8,9: sopB⁺ (S. Typhimurium, humans), lane 10, 11, 12: sopB⁻ (S. Typhimurium,chicken)

Fig. 4

Agarose gel electrophoresis showing an amplification of stn gene (617 bp) in Salmonella isolates from poultry meat and humans. Lane N: negative control (stn ⁻ Salmonella strain), lane L: DNA ladder (100 bp), lane P: Positive control (stn ⁺ Salmonella strain), lane 1: stn ⁻ (S. Derby, duck), lane 2: stn ⁻ (S. Kiel, chicken), lanes 3,7: stn ⁻ (untypable, duck), lane 4: stn ⁻ (S. Rubislaw, chicken), lanes 5,6,10,11,12: stn ⁻ (S. Typhimurium, chicken), lane 8: stn ⁻ (S. Typhimurium, humans), lane 9: stn ⁺ (S. Typhimurium, humans)

Fig. 5

Amplification of conserved regions (5′-CS and 3′-CS) of class 1 integron and integron profiles (IPs) detected in Salmonella isolates from poultry meat and humans. Asterisks represent the bands selected for sequencing. Lane N: negative control, lane L: DNA ladder (100 bp), lane P: Positive control, lane 1: IP I (S. Derby, duck), lane 2: IP II (S. Kiel, chicken), lanes 3,4: IP III (untypable, duck & S. Rubislaw, chicken), lanes 5, 6,10,11, 12: IP IV, V, IX, X (S. Typhimurium, chicken), lanes 8, 9: IPs VII, VIII (S. Typhimurium, humans)

Fig. 6

Amino acid sequence similarities for S. Typhimurium aadA2 gene of the strain under study (S. Typhimurium strain MASR STKT449569) and the reference strains. Dots indicate amino acid positions that are identical to the corresponding S. Typhimurium aadA2 sequence. The glycine 60, isoleucine 61, asparagine 62 and proline 105 in which mutations occur are indicated by the solid bars